The primary objective of this study was the investigation of bioreduction kinetics of hydrophilic spin probes, Tempone, and spin-labeled antibiotic gentamicin by gram-negative bacteria maintained at various oxygen tension, with a special emphasis on the effect of probe penetration rate. This information was used to evaluate the effect of ultrasound on the penetration of hydrophilic compounds, including antibiotics, into P. aeruginosa and E. coli cells through porin channels. Penetration of spin-labeled compounds into the cells was assessed by the reduction rate of the nitroxyl moiety measured by EPR. It was found that probe penetration through the outer cell membrane affected the rate of probe reduction; when the permeability barrier was damaged by cell incubation with EDTA or by powerful insonation above the cavitation threshold, the rate of Tempone reduction by P. aeruginosa cells increased significantly. In contrast, onsonation below the cavitation threshold did not affect the rate of Tempone and spin labeled gentamicin reduction. These results are compared to the effect of insonation on the penetration of hydrophobic compounds into P. aeruginosa cells which proceeds through the phospholipid membrane and is increased by the insonation below the cavitation threshood.